Dengue (DEN) fever and DEN hemorrhagic fever continue to pose significant health problems to human populations in the tropical regions of Asia and the Americas. The principal vector of DEN disease, Aedes aegypti, has spread throughout these tropical regions. The lack of effective vaccines against DEN viruses (serotypes 1-4; Flaviviridae), the collapse of mosquito control programs in many developing countries, and the emergence of pesticide resistant mosquitoes have all had major negative impacts on developing conventional strategies for controlling the disease. The long term goal of this research is to genetically alter the mosquito to eliminate the transmission of DEN viruses. We have previously used a viral expression system derived from the mosquito-borne RNA virus, Sindbis (SIN; Togaviridae) to "cytoplasmically transform" Aedes aegypti vectors with sense and antisense RNAs targeted to the DEN type 2 (DEN-2) antigenome or genome, respectively. These strategies were extremely effective in establishing resistance to DEN-2 virus replication in Aedes aegypti. However, to date RNA mediated interference strategies have been serotype specific. In this proposal, we will use double subgenomic Sindbis (dsSlN) transducing viruses to express and characterize anti-DEN single chain antibodies in Aedes aegypti. We strongly believe that these antibodies will prove to be a new class of agents that will allow us to ablate transmission of all four serotypes of DEN viruses in a single mosquito. We propose to clone from 11 different hybridoma cell lines, single chain antibodies that are reactive to DEN viruses and initially express and characterize these antibodies from prokaryotic expression systems. Once the binding properties and specificities of the single chain antibodies have been determined, the antibodies will be expressed in mosquito cells and adult mosquitoes by the dsSlN transducing viruses. Using established protocols, mosquitoes will be challenged with DEN viruses to determine if anti-DEN virus single chain antibodies can establish intracellular interference to DEN virus replication and ablate transmission of the viruses. Finally, the single chain antibodies will be expressed in transformed mosquito cells to assess the ability of these agents to interfere with DEN virus replication at the DNA level. In toto, this work should give us a significant understanding of a potentially important strategy for ultimately generating transgenic mosquitoes that are resistant to DEN viruses.